Mechanism study of lncRNA RMRP regulating esophageal squamous cell carcinoma through miR-580-3p/ATP13A3 axis

Objective It is well-known that lncRNAs regulate energy metabolism in tumors. This study focused on the action of RMRP on esophageal squamous cell carcinoma (ESCC) cell proliferation, apoptosis, and glycolysis. Methods In the resected ESCC tissues and adjacent tissues from patients, RMRP/miR-580-3p/ATP13A3 expressions were evaluated. ESCC cell proliferation rates and apoptotic rates were measured by CCK-8 and flow cytometry, respectively. Apoptosis related markers were examined by Western blot. Moreover, glucose uptake, lactic acid, and ATP were measured by commercial kits, whereas HK2 and PKM2 were evaluated by Western blot to study ESCC cell glycolysis. Finally, the editing program of RMRP/miR-580-3p/ATP13A3 was translated by luciferase reporter assay and RIP analysis. Results RMRP and ATP13A3 were induced, while miR-580-3p was reduced in their expression in ESCC tissues. Silencing RMRP reduced proliferation, glycolysis, and anti-apoptosis ability of ESCC cells. RMRP sequestered miR-580-3p to target ATP13A3. Silenced ATP13A3 or overexpressed miR-580-3p rescued overexpressed RMRP-mediated promotion of proliferation, glycolysis, and anti-apoptosis of ESCC cells. Conclusion RMRP accelerates ESCC progression through the miR-580-3p/ATP13A3 axis, renewing a reference for lncRNA-based therapies for tumors. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-024-00990-6.


Introduction
Esophageal squamous cell carcinoma (ESCC) is the most common histological subtype of esophageal carcinoma, and its etiology is related to diet, genetic factors, microorganisms, and other environmental factors [1].Multidisciplinary evaluation and treatment have shown survival benefits, such as endoscopic resection for early ESCC, minimally invasive esophagectomy as primary therapy or post-induction chemoradiotherapy, and immunotherapy for metastatic or persistent ESCC cases [2].Increased glycolysis is a characteristic of cancer metabolism and glycolysis is a carcinogenic event [3].Glycolysis is less effective than oxidative phosphorylation in terms of production of adenosine triphosphate (ATP), but cancer cells adapt to this disadvantage by increasing glucose absorption, which in turn increases glycolysis rates [4].Glycolysis not only supports the metabolic needs of tumor cells, but also provides an immune protective ecological niche to activate malignant proliferation, maintenance, and progression [5].Considering the contribution of glycolysis in the tumor microenvironment, targeting glycolysis may be a potential target for therapy [6].
Changes in lncRNA expression and its mutation promote tumor development and metastasis [7].LncRNAs regulate energy metabolism in tumors.Insight into lncRNA-mediated metabolic reprogramming can help identify cellular vulnerabilities to improve tumor diagnosis and treatment [8].Many lncRNAs [9][10][11] have been reported to correlate to malignant properties of ESCC cells, including but not limited to glycolysis.Notably, a recent paper has emphasized the clinical association between lncRNA RNA component of mitochondrial RNA processing endoribonuclease (RMRP) and ESCC patients' poor prognosis and tumor stage [12].Not only in ESCC, but RMRP dysregulation has also been addressed in other cancers and is commonly considered a promoter in tumor malignancy [13][14][15].Nevertheless, more understandings of RMRP-related mechanisms are still lacking in ESCC.
miR-580-3p has been identified to be sponged by RMRP [16].In addition to that, bioinformatics analysis also demonstrated the binding possibility of miR-580-3p and RMRP.In fact, miR-580-3p could be either a tumor inhibitor [17] or a tumor inducer [18] depending on the tumor type.However, it specific property in ESCC lacks scientific investigations until now.Therefore, miR-580-3p, as a downstream molecule of RMRP, was studied in ESCC.
The current research planned to unravel RMRP-mediated proliferation, apoptosis, and glycolysis processes in ESCC and further determined its interplay with miR-580-3p and the downstream target ATP13A3, hoping to provide more evidence for molecule-based targeted therapies for ESCC.

Clinical samples
From April 2017 to March 2021, a total of 53 pairs of ESCC tissues and paracancer normal tissues were gained from The Fourth Hospital of Hebei Medical University.All tissues after identification by two independent pathologists were frozen in liquid nitrogen and stored at − 80 °C.All patients treated with radiation, chemotherapy, or other drugs were removed.The study was approved by the Ethics Committee of The Fourth Hospital of Hebei Medical University (No. 20206HB10).The patient had signed informed consent.

Nuclear-cytoplasmic fractionation and quantitative PCR
Cytoplasmic and nuclear RNA were isolated by PARIS™ Kit (Invitrogen).Trizol reagent (Invitrogen) was employed to gain total RNA which was then processed with reverse transcription to obtain cDNA of lncRNA and mRNA using PrimeScriptT-MII First Strand cDNA Synthesis Kit (Takara, Japan) and that of miRNA using miScript reverse transcription kit (Qiagen, USA).Quantitative PCR was completed using Power SYBR_green PCR master mix (Applied Biosystems) in the ABI 7500 PCR machine.GAPDH and U6 serve as endogenous reference genes. 2 −ΔΔCt was adopted to analyze gene levels.Table 1 exhibits primer sequences used for PCR.

Fluorescence in situ hybridization
Fluorescence In Situ Hybridization (FISH) assay was conducted using the FISH Kit (RiboBio, China) according to the manufacturer's instruction.Briefly, cells were first cultured in 24-well plates for 24 h and then hybridized with 20 μM Cy3labeled U6, 18S and RMRP.Finally, the images were observed with the fluorescence microscope (Leica, Wetzlar, Germany).

Glycolysis measurements
The Glucose Absorption Test Kit (Biovision, CA, USA) measures glucose absorption.The d-lactic acid detection kit and ATP colorimetric/fluorescent detection kit (Biovision) measured the production of lactic acid and ATP, respectively.The corresponding absorbance was detected using a microplate reader.

Analysis of luciferase activity
Wild-type (WT) RMRP and ATP13A3 3′-UTR fragments containing predicted miR-580-3p binding sites were amplified and inserted into pmirGLO vectors (Promega, USA) to establish the reporters RMRP-WT and ATP13A3-WT.GeneArt™ sitedirected mutagenesis PLUS system (A14604; Thermo Fisher Scientific) mutated the putative binding site of miR-580-3p in RMRP and ATP13A3 3' -UTR.The mutant (Mut) RMRP and ATP13A3 3′-UTR were inserted into the pmirGLO vector to construct the reporters RMRP-mut and ATP13A3-mut.KYSE150 cells were co-treated with the reporters and miR-580-3p mimic or mimic NC using Lipofectamine 3000 reagent and collected after 48 h to measure luciferase activity in a dual luciferase reporting system (Promega, Madison, WI, USA) as per protocol.

RIP experiment
RIP testing was guided under the instruction of RIP kit (Millipore, USA).KYSE150 cells were lysed using a RIP lysis buffer and combined with RIP buffers containing magnetic beads conjugated with human anti-AgO2.Protease K was then applied to digest the protein and isolate the immunoprecipitated RNA.RNA expression was detected by quantitative PCR.

Data analysis
At least 3 biological replicates were needed for each experiment.All statistical analyses were performed using Graph-Pad Prism 9.0 software.The student t test analyzed two-group differences, while one-way analysis of variance analyzed multiple-group differences.*P < 0.05 was considered a significant difference.

RMRP expression abundance in ESCC
On the bioinformatics website http:// www.nonco de.org, RMRP was located at chromosome chr9: 35657750-35658018 [-], with a length of 268 bp (Fig. 1A).Quantitative PCR evaluated RMRP expression signature in ESCC and revealed that RMRP in ESCC tissues and cell lines was higher than that in the normal control (Fig. 1B, C).Chi-square test was conducted to evaluate the correlation between the clinical features of ESCC patients with RMRP expression signature.https:// rnasy su.com/ encori/ index.php analysis revealed patients with high RMRP expression possessed poorer survival than patients with low RMRP expression in esophageal carcinoma which was further confirmed by our finding that patients with high RMRP showed worse 5-year survival in ESCC (Fig. 1D, E).
To further elucidate the mechanisms of RMRP, northern blot analysis of RMRP in KYSE150 cells was performed to confirm that RMRP is a non-coding RNA (Fig. 1F).Nuclear-cytoplasmic fractionation assay and FISH assay were conducted to show that RMRP was mainly located in the cytoplasm of KYSE150 cells (Fig. 1G, H).

RMRP deficiency inhibits ESCC proliferation and glycolysis and promotes apoptosis
siRNA of RMRP was treated in KYSE150 cells, leading to the inhibition of RMRP expression in cells (Fig. 2A).CCK-8 assay showed that inhibiting RMRP reduced cell proliferation rate (Fig. 2B), whereas flow cytometry determined its promoting impact on apoptosis rate (Fig. 2C).Moreover, RMRP silenced ESCC cells showed significantly higher expression of proapoptotic protein (cleaved Caspase-3) and significantly lower expression of apoptosis inhibitory protein(Bcl-2) (Fig. 2D).Changes in cellular glycolysis were then assessed.Knocking down RMRP reduced glucose consumption, lactic acid production, and ATP levels in cells (Fig. 2E-G) and inhibited protein expressions of glycolytic proteins HK2 and PKM2 (Fig. 2H).

RMRP targets miR-580-3p
Bioinformatics website starbase predicted potential binding sites between RMRP and miR-580-3p.In the setting of ESCC, quantitative PCR assessed miR-580-3p expression and discovered its low expression pattern in ESCC tissues and cell lines compared with normal controls (Fig. 3A, B).Results of dual luciferase assay demonstrated that miR-580-3p mimic had the ability to reduce the luciferase activity of WT-RMRP (Fig. 3C).Moreover, RIP assay further confirmed that Ago2 was able to enrich miR-580-3p and RMRP in the immunoprecipitates (Fig. 3D).Concerning to miR-580-3p expression mediated by RMRP, quantitative PCR revealed that RMRP deficiency forced miR-580-3p expression in cells (Fig. 3E).

Discussion
About 90% of esophageal cancers are ESCC, which has a poor prognosis and high mortality [19].It is s argued that cell proliferation requires energy, nutrients, and biosynthesis to replicate macromolecular components in cell passages.
Glycolysis is primarily to maintain high levels of glycolic intermediates to support metabolic requirements of cell proliferation [20].Unlike normal healthy cells, cancer cells are more metabolically active, proliferate at a higher rate, and are able to resist cell death pathways such as apoptosis [21].Considering the significance of glycolysis during cancer cell proliferation and apoptosis, this work paid much attention to RMRP's roles in regulating tumor glycolysis to interfere with ESCC progression.As the findings indicated, RMRP exhibited high expression levels in ESCC and indicated a close association with larger tumor size and distal metastasis, as well as poor 5-year survival rates.In addition to that, cell experiments observed that silencing RMRP suppressed proliferation rates, elevated apoptosis rates, lowered glucose consumption, lactic acid production, and ATP levels, and inhibited HK2 and PKM2 protein expression, providing evidence that RMRP prevented proliferative and glycolytic activities and induced apoptosis during ESCC cell growth.Actually, RMRP is considered and identified as an emerging target in carcinogenesis and tumor therapy [22] and is a potent regulator of metabolic reprogramming in cancer [23].Specifically, highly expressed RMRP has been studied in bladder cancer patients and is associated with tumor size, lymph node metastasis, and patients' survival [24].Furthermore, RMRP has been identified as an upregulated lncRNA in non-small cell lung cancer, and it acts mainly on tumor cells in part by forcing cell proliferation [25].Notably, Chen et al. report RMRP expression abundance in colorectal cancer and this abundance is related to an unfavorable prognosis, and moreover, RMRP can facilitate tumor cell proliferation depending on p53 [26].Glycolysis is the backbone of cancer cell metabolism, and cancer cells have evolved various mechanisms to enhance it [33].Evidence swiftly accumulates of lncRNAs influencing glycolysis of cancer cells.In ESCC, researchers reported many lncRNAS have emerged as potent regulators of glycolysis, such as lncRNA G077640 [34], lncRNA-LET [35], lncRNA PTPRG-AS1 [34], et al.Concerning the correlation between RMRP and glycolysis, a report has illustrated that RMRP deficiency contributes to the reduction of glucose uptake, lactic acid, and ATP production in ovarian cancer cells resistant to paclitaxel [16].In the setting of ESCC,

Fig. 2
Fig. 2 RMRP deficiency inhibits ESCC proliferation and glycolysis and promotes apoptosis.RMRP siRNA was transfected into KYSE150 cells.A Quantitative PCR assessed RMRP.B CCK-8 assay measured cell proliferation rate.C Flow cytometry detected cell apoptosis rate.D Western blot evaluated cleaved Caspase-3 and Bcl-2.E-G Commercial kits detected cellular glucose consumption, lactate production, and ATP levels.H Western blot evaluated HK2 and PKM2.Data were expressed as mean ± SD (N = 3).*P < 0.05

Table 2
Abnormally high expression of RMRP in ESCC.A Bioinformatics website http:// www.nonco de.org for RMRP gene information.B Quantitative PCR assessed RMRP in patients' tissues.C Quantitative PCR assessed RMRP in ESCC cell lines and normal esophageal epithelial cells.D Relationship between survival rate and RMRP expression in esophageal carcinoma patients analysed by ENCORI website.E Relationship between survival rate and RMRP expression in ESCC patients.F Northern blot analysis of RMRP expression in KYSE150 cells.G Quantitative PCR assessed RMRP after the fractionation of nuclear and cytoplasmic RNA of KYSE150 cells.H Representative RNA-FISH images showing the subcellular location of RMRP in KYSE150 cells, 18S and U6 were used as cytoplasmic and nuclear markers, respectively (Scale bar, 100 μm).Data were expressed as mean ± SD (N = 3).* P < 0.05 shows that RMRP was highly correlated with tumor size and distal metastasis in ESCC patients.In addition, ENCORI website Fig. 1

Table 2
Correlation analysis of clinicopathological features in patients with RMRP and ESCC